Skip to main content

Thank you for visiting nature.com. You are using a browser version with limited support for CSS. To obtain the best experience, we recommend you use a more up to date browser (or turn off compatibility mode in Internet Explorer). In the meantime, to ensure continued support, we are displaying the site without styles and JavaScript.

Oral application of clozapine-N-oxide using the micropipette-guided drug administration (MDA) method in mouse DREADD systems

Abstract

The designer receptor exclusively activated by designer drugs (DREADD) system is one of the most widely used chemogenetic techniques to modulate the activity of cell populations in the brains of behaving animals. DREADDs are activated by acute or chronic administration of their ligand, clozapine-N-oxide (CNO). There is, however, a current lack of a non-invasive CNO administration technique that can control for drug timing and dosing without inducing substantial distress for the animals. Here, we evaluated whether the recently developed micropipette-guided drug administration (MDA) method, which has been used as a non-invasive and minimally stressful alternative to oral gavages, may be applied to administer CNO orally to activate DREADDs in a dosing- and timing-controlled manner. Unlike standard intraperitoneal injections, administration of vehicle substances via MDA did not elevate plasma levels of the major stress hormone, corticosterone, and did not attenuate exploratory activity in the open field test. At the same time, however, administration of CNO via MDA or intraperitoneally was equally efficient in activating hM3DGq-expressing neurons in the medial prefrontal cortex, as evident by time-dependent increases in mRNA levels of neuronal immediate early genes (cFos, Arc and Zif268) and cFos-immunoreactive neurons. Compared to vehicle given via MDA, oral administration of CNO via MDA was also found to potently increase locomotor activity in mice that express hM3DGq in prefrontal neurons. Taken together, our study confirms the effectiveness of CNO given orally via MDA and provides a novel method for non-stressful, yet well controllable CNO treatments in mouse DREADD systems.

Access options

Rent or Buy article

Get time limited or full article access on ReadCube.

from$8.99

All prices are NET prices.

Fig. 1: The three procedural steps of the MDA method in C57BL6/N mice.
Fig. 2: Effects of VEH administration via intraperitoneal injection or MDA on acute stress response and locomotor activity in the open field test.
Fig. 3: Neuronal activation in the mPFC after CNO administration via IP injection or MDA.
Fig. 4: Effect of chemogenetically induced neuronal activation in the mPFC using the MDA method on spontaneous locomotor activity.

Data availability

The data that support the findings of this study are available from the corresponding author upon reasonable request.

References

  1. 1.

    Roth, B. L. DREADDs for neuroscientists. Neuron 89, 683–694 (2016).

    CAS  PubMed  PubMed Central  Google Scholar 

  2. 2.

    Armbruster, B. N., Li, X., Pausch, M. H., Herlitze, S. & Roth, B. L. Evolving the lock to fit the key to create a family of G protein-coupled receptors potently activated by an inert ligand. Proc. Natl Acad. Sci. U. S. A. 104, 5163–5168 (2007).

    PubMed  PubMed Central  Google Scholar 

  3. 3.

    Whissell, P. D., Tohyama, S. & Martin, L. J. The use of DREADDs to deconstruct behavior. Front. Genet. 7, 70 (2016).

    PubMed  PubMed Central  Google Scholar 

  4. 4.

    Zhan, J., Komal, R., Keenan, W. T., Hattar, S. & Fernandez, D. C. Non-invasive strategies for chronic manipulation of DREADD-controlled neuronal activity. J. Vis. Exp. (150), e59439 (2019).

  5. 5.

    Machholz, E., Mulder, G., Ruiz, C., Corning, B. F. & Pritchett-Corning, K. R. Manual restraint and common compound administration routes in mice and rats. J. Vis. Exp. (67), e2771 (2012).

  6. 6.

    Meijer, M. K., Spruijt, B. M., van Zutphen, L. F. & Baumans, V. Effect of restraint and injection methods on heart rate and body temperature in mice. Lab. Anim. 40, 382–391 (2006).

    CAS  PubMed  PubMed Central  Google Scholar 

  7. 7.

    Stuart, S. A. & Robinson, E. S. Reducing the stress of drug administration: implications for the 3Rs. Sci. Rep. 5, 14288 (2015).

    CAS  PubMed  PubMed Central  Google Scholar 

  8. 8.

    Lewis, R. E., Kunz, A. L. & Bell, R. E. Error of intraperitoneal injections in rats. Lab. Anim. Care 16, 505–509 (1966).

    CAS  PubMed  PubMed Central  Google Scholar 

  9. 9.

    Morton, D. B. et al. Refining procedures for the administration of substances. Report of the BVAAWF/FRAME/RSPCA/UFAW Joint Working Group on Refinement. British Veterinary Association Animal Welfare Foundation/Fund for the Replacement of Animals in Medical Experiments/Royal Society for the Prevention of Cruelty to Animals/Universities Federation for Animal Welfare. Lab. Anim. 35, 1–41 (2001).

    CAS  PubMed  PubMed Central  Google Scholar 

  10. 10.

    Turner, P. V., Brabb, T., Pekow, C. & Vasbinder, M. A. Administration of substances to laboratory animals: routes of administration and factors to consider. J. Am. Assoc. Lab. Anim. Sci. 50, 600–613 (2011).

    CAS  PubMed  PubMed Central  Google Scholar 

  11. 11.

    Fernandez, D. C. et al. Light affects mood and learning through distinct retina-brain pathways. Cell 175, 71–84.e18 (2018).

    CAS  PubMed  PubMed Central  Google Scholar 

  12. 12.

    Urban, D. J. et al. Elucidation of the behavioral program and neuronal network encoded by dorsal raphe serotonergic neurons. Neuropsychopharmacology 41, 1404–1415 (2016).

    CAS  PubMed  PubMed Central  Google Scholar 

  13. 13.

    Jain, S. et al. Chronic activation of a designer G(q)-coupled receptor improves beta cell function. J. Clin. Invest. 123, 1750–1762 (2013).

    CAS  PubMed  PubMed Central  Google Scholar 

  14. 14.

    Scarborough, J. et al. Preclinical validation of the micropipette-guided drug administration (MDA) method in the maternal immune activation model of neurodevelopmental disorders. Brain Behav. Immun. 88, 461–470 (2020).

    CAS  PubMed  PubMed Central  Google Scholar 

  15. 15.

    Alexander, G. M. et al. Remote control of neuronal activity in transgenic mice expressing evolved G protein-coupled receptors. Neuron 63, 27–39 (2009).

    CAS  PubMed  PubMed Central  Google Scholar 

  16. 16.

    Notter, T. et al. Neuronal activity increases translocator protein (TSPO) levels. Mol. Psychiatry Forthcoming (2020).

  17. 17.

    Campbell, E. J. & Marchant, N. J. The use of chemogenetics in behavioural neuroscience: receptor variants, targeting approaches and caveats. Br. J. Pharmacol. 175, 994–1003 (2018).

    CAS  PubMed  PubMed Central  Google Scholar 

  18. 18.

    Jansen van’t Land, C. & Hendriksen, C. F. Change in locomotor activity pattern in mice: a model for recognition of distress? Lab. Anim. 29, 286–293 (1995).

    PubMed  PubMed Central  Google Scholar 

  19. 19.

    Matson, D. J., Broom, D. C. & Cortright, D. N. Locomotor activity in a novel environment as a test of inflammatory pain in rats. Methods Mol. Biol. 617, 67–78 (2010).

    PubMed  PubMed Central  Google Scholar 

  20. 20.

    Gronli, J. et al. Effects of chronic mild stress on sexual behavior, locomotor activity and consumption of sucrose and saccharine solutions. Physiol. Behav. 84, 571–577 (2005).

    CAS  PubMed  PubMed Central  Google Scholar 

  21. 21.

    Shoji, H. & Miyakawa, T. Differential effects of stress exposure via two types of restraint apparatuses on behavior and plasma corticosterone level in inbred male BALB/cAJcl mice. Neuropsychopharmacol. Rep. 40, 73–84 (2020).

    CAS  PubMed  PubMed Central  Google Scholar 

  22. 22.

    Tuli, J. S., Smith, J. A. & Morton, D. B. Stress measurements in mice after transportation. Lab. Anim. 29, 132–138 (1995).

    CAS  PubMed  PubMed Central  Google Scholar 

  23. 23.

    Malisch, J. L. et al. Acute restraint stress alters wheel-running behavior immediately following stress and up to 20 hours later in house mice. Physiol. Biochem. Zool. 89, 546–552 (2016).

    PubMed  PubMed Central  Google Scholar 

  24. 24.

    National Research Council Committee on Recognition and Alleviation of Pain in Laboratory Animals. Recognition and Alleviation of Pain in Laboratory Animals. (National Academies Press (US), Washington, DC, USA, 2009).

  25. 25.

    Belzung, C. & Griebel, G. Measuring normal and pathological anxiety-like behaviour in mice: a review. Behav. Brain Res. 125, 141–149 (2001).

    CAS  PubMed  PubMed Central  Google Scholar 

  26. 26.

    Zimprich, A. et al. A robust and reliable non-invasive test for stress responsivity in mice. Front. Behav. Neurosci. 8, 125 (2014).

    PubMed  PubMed Central  Google Scholar 

  27. 27.

    Salvi, S. S. et al. Acute chemogenetic activation of CamKIIα-positive forebrain excitatory neurons regulates anxiety-like behaviour in mice. Front. Behav. Neurosci. 13, 249 (2019).

    CAS  PubMed  PubMed Central  Google Scholar 

  28. 28.

    Lukas, G., Brindle, S. D. & Greengard, P. The route of absorption of intraperitoneally administered compounds. J. Pharmacol. Exp. Ther. 178, 562–564 (1971).

    CAS  PubMed  PubMed Central  Google Scholar 

  29. 29.

    Gomez, J. L. et al. Chemogenetics revealed: DREADD occupancy and activation via converted clozapine. Science 357, 503–507 (2017).

    CAS  PubMed  PubMed Central  Google Scholar 

  30. 30.

    Fernandez, J. W., Grizzell, J. A., Philpot, R. M. & Wecker, L. Postpartum depression in rats: differences in swim test immobility, sucrose preference and nurturing behaviors. Behav. Brain Res. 272, 75–82 (2014).

    PubMed  PubMed Central  Google Scholar 

  31. 31.

    Wyvell, C. L. & Berridge, K. C. Intra-accumbens amphetamine increases the conditioned incentive salience of sucrose reward: enhancement of reward ‘wanting’ without enhanced ‘liking’ or response reinforcement. J. Neurosci. 20, 8122–8130 (2000).

    CAS  PubMed  PubMed Central  Google Scholar 

  32. 32.

    Donato, F., Jacobsen, R. I., Moser, M. B. & Moser, E. I. Stellate cells drive maturation of the entorhinal-hippocampal circuit. Science 355, eaai8178 (2017).

    PubMed  PubMed Central  Google Scholar 

  33. 33.

    Mahler, S. V. et al. Designer receptors show role for ventral pallidum input to ventral tegmental area in cocaine seeking. Nat. Neurosci. 17, 577–585 (2014).

    CAS  PubMed  PubMed Central  Google Scholar 

  34. 34.

    Lichtenberg, N. T. et al. Basolateral amygdala to orbitofrontal cortex projections enable cue-triggered reward expectations. J. Neurosci. 37, 8374–8384 (2017).

    CAS  PubMed  PubMed Central  Google Scholar 

  35. 35.

    Arakawa, H. Ethological approach to social isolation effects in behavioral studies of laboratory rodents. Behav. Brain Res. 341, 98–108 (2018).

    PubMed  PubMed Central  Google Scholar 

  36. 36.

    Lukkes, J. L., Watt, M. J., Lowry, C. A. & Forster, G. L. Consequences of post-weaning social isolation on anxiety behavior and related neural circuits in rodents. Front. Behav. Neurosci. 3, 18 (2009).

    PubMed  PubMed Central  Google Scholar 

  37. 37.

    Mueller, F. S., Polesel, M., Richetto, J., Meyer, U. & Weber-Stadlbauer, U. Mouse models of maternal immune activation: mind your caging system! Brain Behav. Immun. 73, 643–660 (2018).

    CAS  PubMed  PubMed Central  Google Scholar 

  38. 38.

    Soden, M. E. et al. Genetic isolation of hypothalamic neurons that regulate context-specific male social behavior. Cell Rep. 16, 304–313 (2016).

    CAS  PubMed  PubMed Central  Google Scholar 

  39. 39.

    Farrell, M. S. et al. A Gαs DREADD mouse for selective modulation of cAMP production in striatopallidal neurons. Neuropsychopharmacology 38, 854–862 (2013).

    CAS  PubMed  PubMed Central  Google Scholar 

  40. 40.

    Richetto, J. et al. Genome-wide DNA methylation changes in a mouse model of infection-mediated neurodevelopmental disorders. Biol. Psychiatry 81, 265–276 (2017).

    CAS  PubMed  PubMed Central  Google Scholar 

  41. 41.

    Weber-Stadlbauer, U. et al. Transgenerational transmission and modification of pathological traits induced by prenatal immune activation. Mol. Psychiatry 22, 102–112 (2017).

    CAS  PubMed  PubMed Central  Google Scholar 

  42. 42.

    Notter, T., Panzanelli, P., Pfister, S., Mircsof, D. & Fritschy, J. M. A protocol for concurrent high-quality immunohistochemical and biochemical analyses in adult mouse central nervous system. Eur. J. Neurosci. 39, 165–175 (2014).

    PubMed  PubMed Central  Google Scholar 

  43. 43.

    Livak, K. J. & Schmittgen, T. D. Analysis of relative gene expression data using real-time quantitative PCR and the 2−ΔΔ CT method. Methods 25, 402–408 (2001).

    CAS  Google Scholar 

  44. 44.

    Richetto, J., Polesel, M. & Weber-Stadlbauer, U. Effects of light and dark phase testing on the investigation of behavioural paradigms in mice: relevance for behavioural neuroscience. Pharmacol. Biochem. Behav. 178, 19–29 (2019).

    CAS  PubMed  PubMed Central  Google Scholar 

  45. 45.

    Notter, T. et al. Translational evaluation of translocator protein as a marker of neuroinflammation in schizophrenia. Mol. Psychiatry 23, 323–334 (2018).

    CAS  PubMed  PubMed Central  Google Scholar 

Download references

Acknowledgements

This study was supported by a Postdoc Mobility grant (grant no. P2ZHP3_174868) awarded to T.N. by the Swiss National Science Foundation. Additional financial support was received from the Swiss National Science Foundation (grant no. 310030_188524 awarded to U.M.; grant no. PZ00P3_18009/1 awarded to J.R.). We would like to thank the Hodge Foundation UK, as well as the Viral Vector Facility (VVF) of the Neuroscience Center Zurich (ZNZ). Imaging was performed with equipment maintained by the Center for Microscopy and Image Analysis, University of Zurich.

Author information

Affiliations

Authors

Contributions

S.M.S. designed and performed research, analyzed data and contributed to the preparation of the manuscript. F.S.M., J.S., J.R. and U.W.-S. performed research. U.M. designed research and contributed to the preparation of the manuscript. T.N. designed and performed research, analyzed data and wrote the manuscript.

Corresponding author

Correspondence to Tina Notter.

Ethics declarations

Competing interests

Unrelated to the present study, U.M. has received financial support from Boehringer Ingelheim Pharma GmbH & Co. and from Wren Therapeutics Ltd. All authors declare no competing interests.

Additional information

Publisher’s note Springer Nature remains neutral with regard to jurisdictional claims in published maps and institutional affiliations.

Extended data

Extended Data Fig. 1 Immediate early gene expression in the mPFC after CNO (1 mg/kg) or VEH administration via IP injection or MDA in the absence of the modified human muscarinic M3 G-protein-coupled receptor (DREADD).

The scatter bar blots represent mRNA levels of cFos, Arc and Zif268 in the mPFC of adult (12-week-old) C57Bl6/N mice 60 min after treatment with VEH or CNO given via MDA or IP injections; n(VEH/IP) = 7, n(CNO/IP) = 7, n(VEH/MDA) = 7, n(CNO/MDA) = 7. Each dot in the scatter bar plot represents an individual animal, and error bars represent s.e.m.

Supplementary information

Rights and permissions

Reprints and Permissions

About this article

Verify currency and authenticity via CrossMark

Cite this article

Schalbetter, S.M., Mueller, F.S., Scarborough, J. et al. Oral application of clozapine-N-oxide using the micropipette-guided drug administration (MDA) method in mouse DREADD systems. Lab Anim 50, 69–75 (2021). https://doi.org/10.1038/s41684-021-00723-0

Download citation

Search

Quick links

Nature Briefing

Sign up for the Nature Briefing newsletter — what matters in science, free to your inbox daily.

Get the most important science stories of the day, free in your inbox. Sign up for Nature Briefing